Neuropeptide Y and
corticotropin-releasing factor
bi-directionally
modulate inhibitory synaptic
transmission in the bed nucleus
of the stria terminalis
Thomas L. Kash, Danny G.
Winder
Introduction
Neuropeptides (NPY) = potent
neuromodulators in the CNS
Involved in reward pathway
mediated via G-protein coupled
receptors
released in a frequency dependent
fashion
longer half-life of activity after release
Introduction
Corticotropin Releasing Factor (CRF)
involved in stress response
Mediated through the hypothalamus
and the amygdala
Both pathways converge at the BNST
Introduction
Life = Stress
Repeated or severe stressors can
produce behaviors such as posttraumatic stress disorder and
generalized anxiety disorder
BNST provides a substrate for
interaction of CRF and NPY in
regulating stress and anxiety
Introduction
NPY
Anxiety ↓
Reward pathway
CRF
Anxiety ↑
Stress response
BNST acts as a scale to create a
balance of CRF and NPY
Neves S, Ram P, Iyengar R. G protein pathways. Science 296, 1636-1639 (2002)
Introduction
BNST expresses both NPY/YRs and
CRF/CRFRs
CeA releases CRF and GABA to the
BNST
Both CRF and NPY modulate
GABAergic transmissions
Introduction
Study GABAergic influence on
ventrolateral region of the BNST
vlBNST projects to the VTA (reward)
and the PVN (stress)
Introduction
IPSC= inhibitory post-synaptic current
m = miniature
e = evoked
Methods
Male C57B1/6J mice
Decapitated mice and placed brain in icecold sucrose aCSF
Slices 300um thick
Rostral slices contained anterior BNST
Stored in heated, oxygenated container w/
aCSF
Transferred to submerged recording chamber
Heated, O2 aCSF for 1h ā experiments
Methods
Slices in chamber and neurons of vlBNST
visualized w/ infrared video microscopy
Analyzed eIPSC & EPSC
Electrodes filled w/ pH 7.2
Twisted nichrome wire
Placed in vlBNST
Cells held @ -50mV & GABAAR-mediated
IPSCs evoked @ 0.2 Hz by fiber stim w/
bipolar electrodes
Methods
GABAA-IPSCs (& EPSC) isolated
3mM kynurenic acid (& 25uM picrotoxin) =
block AMPA & NMDA receptor-dependent
postsynaptic currents
1uM CGP 55845 = block GABABR
Signals acquired via Multiclamp 700B
amplifier
Input and series resistance continuously
monitored
Methods
eIPSC → measured peak amplitude of
synaptic response normalized to baseline
Baseline period = 2 min period
immediately preceding drug
Value is 2 min avg 15 min p neuropeptide
B
0
3
*
peptide
5
10
15
20
22
Methods
mIPSC analysis
GABAAR-mediated IPSCs isolated → added
0.5uM TTX
recorded in 120s episodes
Ca2+ influx on NPY → 100uM Cd2+ + aCSF
Amplitude and frequency determined from
120s recording w/ cells held @ -70mV
Multi-clamp
Methods
All drugs applied via bath
All peptides used were dissolved in dH2O
to 0.1mM conc
Some stored @ -20°C
Results
NPY and CRF influence on inhibitory
synaptic transmission in vlBNST
Whole-cell voltage clamp
Local stimulation produced eIPSC
from GABAARs
SR95531= GABAAR antagonist blocked
response
Results
TTX elicited spontaneous mIPSCs
GABAzine = GABAAR antagonist
mIPSCs blocked
GABAAR mediated
Results
NPY depresses GABA through Y2R
Baseline recordings revealed
decreased peak amplitude of eIPSC
Observed in all cells
NPY-induced depression =
concentration dependent
Results
NPY13-36 = Y2R agonist
 ↓ peak amplitude
[Pro34]-NPY = Y1R agonist &
[D-Trp32]-NPY = Y5R agonist
No change
Results
Antagonist testing was preceded by
an exposure to NPY (10-15min)
Agonist and antagonist co-applied
(5min)
NONE had significant effects on
eIPSC without agonist
Results
Non-peptide Y2R antagonist blocked
NPY actions
Peptide Y1R antagonist had no effect
Non-peptide Y5R antagonist had no
effect
Y2Rs activated NPY-induced
eIPSC depression
Results
Paired Pulse Ratio experiments
Pair of eIPSCs w/ 50ms between
Ratio of amplitudes determined
NPY ↑ PPR of eIPSCs
Suggest ↓ release of GABA
NPY ↓ frequency but not amplitude
Results
Presynaptic inhibition of NT release
Modulate Ca2+ entry
Regulate release machinery
At CNS synapse basal mIPSC freq ↓
by using Cd2+ to block Vg Ca2+
channels
Cd2+ + NPY = restores mIPSCs
NPY inhibits GABA via Y2R
regulation of Ca2+ influx
Results
5 min bath of 1um CRF sig ↑ peak
amp of eIPSC
1um Urocortin (CRFR agonist) →
similar results
CRF results were concentration
dependent [100nM vs 10nM]
CRF antagonist had no sig effects on
eIPSC in absence of agonist
Results
Non-peptide CRFR1 antagonist (NBI
27914) blocked both CRF and Ucn I
actions
Peptide CRFR2 antagonist (antiSauvagine-30) had no sig effect
Ucn I enhanced eIPSCs in CRFR2
knockout mice
So, CRF/Ucn I induced enhancement
of eIPSC is d/t CRFR1 activation
Results
CRF
did not alter PPR or mIPSC kinetics
no effect on freq of mIPSCs
Mean amplitude ↑↑
Shifted cum. amplitude curve to the
right
CRF enhanced GABAergic
postsynaptic transmission
Change in IPSCs d/t non-specific
enhancement via synaptic excitability
Discussion
 NPY suppresses GABAergic
transmission in vlBNST via Y2R:
1. NPY effect mimicked by NPY13-36,
not by [Pro34]-NPY (Y1 agonist) or [DTrp32]-NPY (Y5 agonist)
2. NPY antagonized by Y2R antagonist
(BIEE 0246), not by BVD-10 (Y1R
antagonist) or L-152804 (Y5R
antagonist)
Discussion
Data consistent w/ NPY actions in
thalamus & PVN and Y2R expression
in BNST
Y2R ↓ GABA release:
1.NPY ↑ PPR of eIPSCs → ↓ release
probability
2.NPY ↓ mIPSC frequency not amplitude
3.Cd+2 effect → NPY via Y2R inhibit
GABA via presynaptic Ca+2 influx
Discussion
NPY via Y2R → heterocepter on
GABAergic terminals
Which YR is activated determines the
behavioral outcome
Y1R and maybe Y5R → NPY anxiolytic
response
Y2R → anxiogenic response
But, Y2R activation in LC → NPY
anxiolytic effects
Discussion
Suppose region-specific activation of
YR subtypes will evoke distinct
behavioral phenotypes, OR…
Autoreceptor-like functions are
anxiolytic
Discussion
Based on evidence that inhibitory
projections from the vlBNST contact
PVN neurons:
↓ GABAergic input to vlBNST →
↑ GABAergic output to PVN →
↓ stress response
Discussion
In the vlBNST, CRF and Ucn I
enhance GABAergic transmission via
CRFR1:
1.CRF & Ucn I antagonized by NBI 27914
(CRFR1), not AS 30 (CRFR2)
2.Ucn I effects observed in mice lacking
CRFR2
3.CRFR1 enhance postsynaptic GABAA-R
response
Discussion
CRF ↑ mIPSC amplitude not
frequency or PPR
Glutamatergic transmission in vlBNST
not affected by Ucn I
Presynaptic CRFR1 mediates
GABAergic transmission in CeA
Altering mIPSC frequency modulates
GABA release → now amplitude as
well
Discussion
CRFR1 mediates CRF anxiogenic
effects in the BNST
CRF enhancing GABAergic
transmission in BNST could
potentially reactivate the PVN →
stress response